Insights into anterograde trafficking and pharmacological chaperoning of Cannabinoid Receptor 2 (CB ₂ )

Cannabinoid Receptor 2 (CB ₂ ) is a promising therapeutic target for modulating inflammation. Canonical signalling responses to receptor ligands are critically dependent on cell surface receptor expression. However, it is also now appreciated that intracellular G protein-coupled receptors can contribute to signalling responses and influence functional outcomes. Therefore, understanding how the subcellular distribution of receptors is controlled is also highly pertinent. CB ₂ is observed to be expressed at the cell surface as well as having a considerable proportion expressed intracellularly. Despite this distribution being well established, little is known about the regulation of CB ₂ anterograde trafficking and subcellular distribution. We report that sustained stimulation with a range of CB ₂ agonists concurrently internalises cell surface human CB ₂ and stimulates a distinct population of CB ₂ to be delivered to the cell surface, at various expression levels. We present evidence that the ligand-stimulated anterograde trafficking is a result of CB ₂ agonists, as well as inverse agonists, acting as pharmacological chaperones. We also report that a di-lysine (KK) motif in the CB ₂ C-terminal tail is required for basal delivery to the cell surface. Corroborating the hypothesis that CB ₂ ligands can act as pharmacological chaperones, sustained CB ₂ ligand stimulation induces cell surface expression of the mutated receptor and alters maturation states as measured by western blotting. Our finding that CB ₂ ligands can unconventionally manipulate CB ₂ cell surface expression may well have important implications for optimal design of CB ₂ -targeted therapeutics.